Animals and Cell culture
6-8 weeks ALB/C nude male mice (the Experimental Animal Center of Xi’an Jiaotong University, Xi’an, China) were used for all in vivo experiments. The methods were carried out in accordance with the approved guidelines of the regional authorities according to China animal-care regulations. K562 cells were cultured in IMDM containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin. JLTRG and H9 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin. BaF3 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 U/mL streptomycin, and 2 ng/mL murine IL-3 (R&D Systems, Inc., Minneapolis, MN, USA). Cells were grown at 37 °C in a humidified atmosphere with 5% CO2.
Human blood samples
Nine peripheral blood (PB) samples of patients were presented from Prof. Yanhong Ji of Xi’an Jiaotong University and nine PB samples of normal subjects are all from of Xi’an Jiaotong University Health Science Center. Mononuclear cells (MCs) were isolated by Ficoll-Paque (GE Healthcare, Waukesha, WI, USA) density gradient centrifugation. Samples were viably frozen in FBS containing 10% DMSO, stored in liquid nitrogen, and later thawed for future analysis. The patients’ consent was obtained in accordance with the Declaration of Helsinki. The participants provided written informed consent to take part in the study. The study protocol was approved by the Biomedical Ethics Committee of Xi'an Jiaotong University Health Science Center (Project No. 2019-966).
Apparatus and chromatographic conditions
Chromatographic analysis was performed on a Shimadzu LC-20A apparatus (Shimadzu, Kyoto, Japan). The data were acquired by using the LC solution software (Shimadzu, Kyoto, Japan). Silica gel (ZEX-II, 5 μm, 100 mesh) was from Qingdao Meigao Chemical Co. Ltd (Qingdao, China). The detection wavelength for the drugs was 247 nm. Mobile phase buffer for chromatography study was 20 mM phosphate buffer (pH 7.4). Flow rate was 0.2 mL/min and temperature was 37 °C.
Cell proliferation assay
Exponential-phase cells were seeded into 96-well plates at a density of 2×104 cells/well in complete medium. After 24 h, cells were treated with drugs at indicated concentrations and incubated for 48 h. Fresh medium containing 20 μL MTT solution (5 mg/mL) were then added to each well. Then plates were incubated for another 4 h at 37 °C. After removing the medium, 150 μL DMSO was added to each well. The absorbance was recorded at 490 nm in the micro-plate reader and the inhibition ratio (I%) was calculated.
Flow cytometric analysis of apoptosis
Exponential-phase cells were treated with vandetanib for 48 h, the cells were then collected, washed, and resuspended in PBS. The apoptotic cell death rate was examined with Annexin V-FITC and PI double staining using the Annexin V-FITC apoptosis detection kit according to the manufacturer’s instructions. After staining the cells with Annexin V-FITC/PI, flow cytometric analysis was performed and data was analyzed using Cell Quest software.
Western blot analysis
Proteins were extracted using RIPA lysis buffer containing protease and phosphatase inhibitor cocktail on ice. The lysates were centrifuged at 12,500×g at 4 °C for 10 min. An equivalent amount of protein was resolved on a 10% SDS-PAGE gel and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with Tris-buffered saline containing 0.05% Tween-20 (TBST) and 5% low-fat powdered milk for 1 h. The blot was then incubated with the primary antibody overnight at 4 °C. After washing with TBST for 10 min three times, the blot was incubated with a secondary antibody for 1 h at 37 °C. The blot was washed three times with TBST before being exposed to the SuperSignal West Dura Extended Duration substrate. Band intensity was quantified by densitometric analyses using a densitometer.
All of the enzymatic reactions were conducted at 30 °C for 40 min. The 50 µL reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/mL BSA, 1 mM DTT, 10 µM ATP, kinase (50 enzyme used/reaction) and the enzyme substrate (0.2 mg/mL Poly (Glu, Tyr)). The compounds were diluted in 10% DMSO and 5 µL of the dilution was added to a 50 µL reaction so that the final concentration of DMSO is 1% in all of reactions. The assay was performed using Kinase-Glo Plus luminescence kinase assay kit. It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity.
RNA extraction and RT-PCR
Total RNA was extracted using the RNAfast200 kit according to the manufacturer’s protocol. The RT-PCR was performed using PrimeScript RT Master Mix Perfect Real Time kit. Real-time PCR was performed using SYBR® Premix Ex TaqTM II and a Thermal Cycle Dice Real time system. The primer sequences were as following:
Human GAPDH forward primer: 5'-GCACCGTCAAGGCTGAGAAC-3'
Human GAPDH reverse primer: 5'-TGGTGAAGACGCCAGTGGA-3'
Human EphB4 forward primer: 5'- CAGGAACATCACAGCCAGAC-3'
Human EphB4 reverse primer: 5'- CAGGACCAGGACCACACC-3'
Mouse GAPDH forward primer: 5'-AGGTTGTCTCCTGCGACTTCA-3'
Mouse GAPDH reverse primer: 5'-TGGTCCAGGGTTTCTTACTCC-3'
Mouse EphB4 forward primer: 5'-TATGCCACGATACGCTTCAC-3'
Mouse EphB4 reverse primer: 5'-CGCTTCCGAGTCAGATGTTC-3'
Melt curve analysis was performed at the end of each PCR to confirm the specificity of the PCR product. Threshold cycle (Ct) values of EphB4 in each sample were normalized with the GAPDH expression. The ratio of EphB4 versus the corresponding GAPDH of each sample was determined on the basis of the equation EphB4/GAPDH = 2Ct(GAPDH) - Ct(EphB4).
EphB4 plasmid extraction was performed according to the instructions of Endo-free Plasmid DNA Mini Kit I (OMEGA). K562 cells were seeded into 6-well plates at the density of 2×105 per well. After 24 h, transfection of EphB4 plasmid into K562 cells was performed using Lipofectamine 2000 reagent, according to the manufacturer’s instructions. The ratio of Lipofectamine 2000 (μL) to EphB4 plasmid (μg) was 2:1. The EphB4 expression was determined by RT-PCR to detect mRNA expression and western blot to detect protein expression.
A double-stranded siRNA against EphB4 and nonspecific siRNA (control siRNA) were from Shanghai GenePharma Co., Ltd. (Shanghai, China). The target sequence of EphB4 oligo selected 5'-CCUUUGAGGUCACUGCAUUTT-3' (sense) and 5'-AAUGCAGUGACCUCAAAGGTT-3' (antisense). The recombinant adenovirus was obtained from Vector Builder (Conrad, Texas). Transfection of K562 cells with adenovirus was performed in 10×10 cm cell culture dish with 5×106 cells/dish in 12 mL IMDM medium containing 10% FBS. 24 h later, adenovirus was added to the dishes at a MOI of 6 and the medium was changed to complete medium and the transfected K562 cells were harvested after 6 days of incubation. The EphB4 expression was determined western blot to detect protein expression.
In this study, each column was equilibrated with the pH 7.4 mobile phase buffer. The mobile phase was then switched to a solution that contained a known concentration of the tested drug in mobile buffer. Each drug solution was continuously applied to the column until a breakthrough curve with a level plateau was produced. The system was later switched back to the mobile phase buffer to elute the retained analyte from the column.
The KD value is investigated by using frontal analysis with the following equation :
Where mLapp represents the moles of analyte required to reach the mean point of the breakthrough curve at a given molar concentration of the applied analyte. [A], KD, mL are the molar concentration of applied analyte, the dissociation equilibrium constants for the analyte at binding sites, and the total moles of binding sites in the column, respectively.
Surface plasmon resonance (SPR) analysis
For the SPR analysis, EphB4 protein was prepared to 50 µg/mL by using PBS buffer. The stock solution of the tested drug was 0.02 M dissolved in DMSO. Working solution of tested drugs was diluted into 20 µM, 10 µM, 5 µM, 1 µM using PBS buffer. Firstly, an NTA chip was installed in the OpenSPRTM instrument and was pumped by using PBS buffer at maximum flow rate. After reaching the signal baseline, 80% isopropanol was injected from the injection port, and the control valve was transferred into “inject”. After running 10s, the control valve was back to “load” for bubble exhaust. When the signal reaches at baseline, the flow rate of running buffer (PBS) was adjusted to 20 µL/min. Then imidazole solution (250 µL) was used to initialize the sensor surface for 3 times, and NiCl2 solution (40 mM, 250 µL) was injected to charge the NTA. Afterwards, His labeled EphB4 (50 µg/mL, 250 µL) dissolved running buffer was injected to interact for 5 min. The activity of the ligand and the maximum surface binding force were confirmed by injecting a high concentration of the tested drug. Finally, increase HCL solution (PH=2, 150 µL/min) was used to remove the sample in the system.
Structure preparation for molecular dynamics simulations
The starting structure for the docking and molecular dynamics simulation of EphB4 was taken from the Protein Data Bank (PDB ID: 3ZEW) , and the structure of vandetanib was obtained from the PubChem (PubChem CID: 3081361). The complex structure of EphB4 and vandetanib was obtained by molecular docking. Docking calculations was carried out using the AutoDock 4.2.
Molecular dynamics (MD) simulations
The MD simulations were performed using the AMBER package version 16. To prepare the topology and coordinate files, the AMBER ff03 all-atom force fields  was used for the protein atoms, and the antechamber module of AMBER Tools was used to assign GAFF  parameters for vandetanib. All MD simulations were carried out by applying periodic boundary conditions in TIP3P water model with a margin of at least 12 Å from any edge of the box to any atom of the solute molecules. The counter ions Na+ were added to neutralize the solvated systems. A 3000-step minimization (steps 1-1000 using conjugated gradient followed by 2000 steps steepest decent) was first carried out using SANDER module. After minimization, a 500 ps constant volume and constant temperature (NVT) simulation was performed to raise the temperature of the system to 298 K while constraining backbone atoms with a 5 kcal/mol/Å2 force constant with reference to the structures. A second 200 ps constant pressure and constant temperature (NPT) simulation at 298 K was performed while constraining backbone atoms with a 2 kcal/mol/Å2 force constant with reference to the structures. The system was then equilibrated for 1 ns at 298K without any constraints. The system is then ready for the 100 ns production run. All the 100 ns MD simulations were in the isobaric isothermal (NPT, T = 298 K and P = 1 atm) ensemble. The SHAKE algorithm was used to fix bonds involving hydrogen. The PME method  was used and the non-bonded cutoff distance was set at 10 Å. The time step was set to 2 fs.
K562 cells were plated into 6-well plates one day prior to treatment with vandetanib (0 or 1.56 μM) for 48 h. Total RNA was isolated using Trizol. Microarray analysis and data processing were done by Gminix (Shanghai, China).
pX459 v2.0 (Fig. S1) was used for sgRNAs and CAS9 gene expression. sgRNAs for EphB4 knockout and point mutation were online designed at http://crispr.mit.edu/. ssODNs for target mutation were designed according to Zhang’s protocol . Oligos used in this article were listed in Table S1.
Construction of K562 EphB4-knockout cell line by CRISPR Cas9
K562 cells were seeded and cultured in 60×15 mm dishes with IMDM medium containing 10% fetal bovine serum (ExCell Biology). Once the cell confluence reached 70-80%, the medium was replaced by IMDM antibiotic-free and serum-reduced medium for 24 h before transfection. By using the ExFect 2000 transfection reagent (Vazyme), the cells were transfected with the construct containing both EphB4 sgRNA and Cas9 protein-coding sequences. The stable EphB4-knockout cells were then harvested after 2 weeks of puromycin (12 µg/mL) treatment.
Generation of EphB4 site-directed mutagenesis cell line
K562 EphB4-knockout cells were seeded and cultured in 60×15 mm dishes to reach 70-80% of cell confluence. The medium was then replaced by an IMDM antibiotic-free and serum-reduced medium for 24 h before transfection. Five mutagenesis construct EphB4-M1, EphB4-M2, EphB4-M3, EphB4-M4, EphB4-M5 were transfected into K562 EphB4-knockout cells by using ExFect 2000 transfection reagent (Vazyme) individually. M1 represent point mutation I14A and G15A, M2 represent point mutation M83A, N85A and G86A, M3 represent point mutation A87Del, M4 represent point mutation S90A and F91A, M5 represent point mutation R93A and L94A. Site-directed mutagenesis cells were then harvested after two days of incubation.
In vivo therapeutic study
In order to evaluate the effect of EphB4 in K562 tumor formation, ALB/C nude mice (4-6 weeks) were injected subcutaneously with wild type K562 cells and stable EphB4 knockdown K562 cells at a density of 2×107 cells (diluted in a mixture containing 100 μL PBS and 100 μL high-concentration matrix) per mouse into the right flanks. After 2 weeks, the mice were euthanized and tumor weight was monitered.
For the in vivo therapeutic study, ALB/C nude mice (4-6 weeks) were injected subcutaneously with wild type K562 cells at a density of 2×107 cells (diluted in a mixture containing 100 μL PBS and 100 μL high-concentration matrix) per mouse into the right flanks. When the tumor volume reached about 80-100 mm3, mice were randomly grouped (n=3) and treated with sodium carboxymethylcellulose or vandetanib (40 mg/kg) every day by intragastric administration. Tumor volumes were calculated every 2 days until day 14 according to the formula: (L*W2)/2 (L and W are the long and short tumor diameters). And in all the period of treatment, body weight was monitored.
In order to evaluate the synergetic action of vandetanib on imatinib-resistant K562 tumor, ALB/C nude mice (4-6 weeks) were injected subcutaneously with imatinib-resistant K562 cells at a density of 2×107 cells (diluted in a mixture containing 100 μL PBS and 100 μL high-concentration matrix) per mouse into the right flanks. When the tumor volume reached about 80-100 mm3, mice were randomly grouped (n=3) and treated with sodium carboxymethylcellulose, vandetanib alone (40 mg/kg), imatinib alone (100 mg/kg), and combination treatment (vandetanib alone (40 mg/kg) plus imatinib (100 mg/kg) ) every day by intragastric administration. Tumor volumes were calculated every 2 days until day 14 according to the formula: (L*W2)/2 (L and W are the long and short tumor diameters). And in all the period of treatment, body weight was monitored.
After completing the in vivo experiment, tumor tissues were collected. Some tumor tissues were collected in -80 °C for the following western blotting. The other tumor tissues were fixed in 4% paraformaldehyde, and then embedded in paraffin for immunohistochemical analysis.
For immunochemistry assay, the paraffin-embedded tumor tissues were sectioned into slices at 5 μm using an HM325 Rotary Microtome. Then the slices were deparaffinized in xylene and hydrated in descending grades of alcohol. Afterwards, the sections were pretreated with 0.01 mol/L citrate-buffered saline (pH 6.0) and quenched with 0.3% (v/v) hydrogen peroxide for endogenous peroxidase activity. Then, the sections were washed twice in distilled water for 5 min each and incubated with diluted normal blocking serum at room temperature for 1 h. After that, the sections were incubated with primary antibody diluted in blocking buffer at 4˚C overnight and washed with PBS for three times before incubating with secondary antibody for 20 min at 95 °C. Then the tissues were washed with PBS and S-A/HRP working solution. The tissues were incubated for 1 min in diaminobenzidine tetra-hydrochloride to develop the peroxidase labeling. Finally, counterstaining was conducted with hematoxylin for imaging.
All values are expressed as the mean ± standard error of means (SEM). Three independent experiments were conducted for each assay. Data were compared using the t test (SPSS statistical package 16.0, Chicago, IL). A P< 0.05 is considered to be statistically significant.